Myotonic dystrophy (DM1) is the most common adult onset muscular dystrophy in humans. Currently, there is no cure or an FDA approved drug for DM1 and related diseases. The molecular basis of DM1 is the expansion of a CTG-repeat sequence in the 3'untranslated region of the protein kinase gene, DMPK. This defect results in the expression of mutant DMPK RNAs encoding expanded CUG repeats (CUGexp) that form large intra nuclear RNA-protein complexes or foci. Expression of CUGexp RNAs leads to abnormal RNA splicing, which in turn has been linked to the development of key features of DM1 pathology. We hypothesize that small molecules that degrade or disperse CUGexp RNAs in DM1 cells can re-establish normal splice patterns and reverse DM1 pathology. To test this hypothesis, we developed a primary HTS and a secondary hit validation assay to identify small-molecules that alter the biology of CUGexp RNAs without affecting the normal transcript. Our in house library was developed using a robust machine learning chemoinformatics platform and consists of 40,000 highly diverse small-molecules representing a library of over a million compounds. An initial screen of 2,500 small molecules from this library resulted in the identification of a potent lead compound, MDI16, which reverses aberrant RNA splice patterns in both DM1 patient myoblasts and in the HSALR mouse model for DM1. In a concerted effort to identify other potent lead compounds we propose the following: Aim 1. Implement primary HTS and the secondary hit validation assay in the MLPCN center. Aim 2. Test hits in tertiary cell-based assays to identify highly potent molecules that reverse four key cellular DM1 phenotypes. Aim 3. Characterize the selectivity and toxicity of lead compounds and identify their mechanism of action at the cellular level using a set of cell-based assays developed in the lab. Aim 4. In conjunction with the MLPCN center, we will refine the chemical structure of lead compounds reiteratively to optimize pharmacological properties and establish structure-activity relationships. PUBLIC HEALTH RELEVANCE: Lay Summary Myotonic dystrophy type 1 is a neuromuscular disorder for which there is no treatment or cure. Over the past few years exciting strides in our understanding of the mechanistic basis of this disorder have been made. Thus the field is poised to make a major breakthrough and develop a drug for this disorder. We have developed a sensitive high throughput screen (HTS) to identify compounds that cure or ameliorate pathologies associated with myotonic dystrophy. Currently we have discovered potent molecules, which rescue DM1 pathology in both DM1 patient myoblasts and in DM1 mouse models. As our screens have been proven to identify potent molecules that rescue DM1 pathology, in this application we propose to identify other lead compounds by screening the MLPCN chemical library with our HTS. Identification of multiple leads will greatly improve the probability of a small molecule therapy for DM1.